Protein biochemistry

fluorescence emission is proportional to the amount of template. The advantage of the TaqMan qPCR method is the usage of differently labelled probes which allow the amplification of several genes in the same sample.

For the relative quantification of parasites in the infected host cells (section 2.2.7), the actin genes of Leishmania spp. and mice (macrophage, host cell), respectively were amplified using primer and probe sets specific for the particular gene (section 2.1.6) (Bifeld et al., submitted). The probe, specific for the murine actin gene was labelled with the Cy5 fluorochrome and the parasite’s actin gene-specific probe was labelled with the FAM fluorochrome.

Genomic DNA was isolated from BMMs (section 2.2.7) and used as a template for the TaqMan qPCR. The qPCR was performed using the KAPA probe fast qPCR kit master mix (VWR company) following the manufacturer’s instructions. Primers were applied to the master mix at a final concentration of 300 mM each. Gene-specific probes were added to the master mix at a final concentration of 200 nM. The template volume did not exceed 10% of the final reaction volume, which was adjusted with nucleic acid-free ddH2O to 20 µL and its amount did not exceed 250 ng.

A one step PCR was performed on a Rotor-Gene^TM Instrument with the following cycling program:

The cycle threshold (Ct) values from the exponential amplification phase were used for the relative quantification of the parasite load in the experimental samples. The Ct values from the parasites actin gene amplification were subtracted from the Ct values from the mouse actin gene amplification in the same sample (ΔCt-method). The normalised values are used to calculate the amplification difference between the sample (e. g. drug treated cells) and the control (e g. non treated cells) (ΔΔCT=ΔCtsample-ΔCtcontrol). The relative parasite load is then expressed by R (ratio)= 2^-ΔΔCt. The significance was calculated by the paired student’s t-test.

length. The induction with 0.4 mM IPTG started at OD600nm between 0.05-0.1 (approximately after 4 hours) and the flasks were incubated in a shaker at 37°C (1 mL of the medium was taken before inducing, pre-induction sample). Two hours after induction, a 1 mL aliquot of the culture was taken to be measured in the spectrophotometer (post-induction sample) and the rest of the cells were harvested at 5,000 ⨉ g for 20 minutes (4°C) (JA 10 rotor, Beckman Coulter). The supernatant was discarded and the pellet was resuspended in 20 mL of PBS and transferred to a 50 mL reaction tube. The samples were centrifuged at 3,220 ⨉ g for 20 minutes at 4°C (JA 12 rotor, Beckman Coulter). The supernatant was discarded and the pellet frozen at -70°C. The pellet was resuspended in 20 mL buffer 1 (20 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole) and sonicated for 20 seconds (50 W) with a 10 seconds break, repeated 6 times. The samples were centrifuged at 10,330 ⨉ g for 30 minutes at 4°C and the supernatant was transferred to a new tube and frozen at -70°C (previously an aliquot of 50 µL was taken as post-induced soluble protein). The pellet was resuspended in 20 mL of buffer 2 (20 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, 8 M urea) and left for 1 hour in a roller shaker at 4°C. Then, the samples were centrifuged at 10,330 ⨉ g for 30 minutes at 4°C. A 1 mL aliquot was taken as induced protein-inclusion bodies.

SDS PAGE was performed to detect whether (His)10-tagged ARM56 was expressed after induction and in which fraction it was. The fractions of interest were pooled for purification.

- Protein purification

The fractions with the recombinant protein were subjected to a refolding assay. For this, 1 L of pre-cooled buffer B5 (20 mM Tris-HCl pH 8.0; 0.5 M KCl, 5 mM imidazole) was transferred to a measurement cylinder with a magnetic stir bar and kept at 4°C. The sample was added drop wise (sample in the 8 M urea solution) to the buffer B5. The (His)10-tagged ARM56 recombinant protein was purified by metal chelate affinity chromatography in a 10 mL matrix of beads charged with NiSO4 (Novagen His-Bind) in a 50 mL column body. The column was equilibrated with two volumes of 5 mL of buffer B5 with a flow rate of 0.5 mL min^-1. After adding the volume to the column, it was washed with 10 mL buffer B5 followed by washing with 5 mL buffer B100 (20 mM Tris-HCl pH 8.0; 0.5 M KCl, 100 mM imidazole) and finally eluted with 10 mL buffer B1000 (20 mM Tris-HCl pH 8;

0.5 M KCl, 1 M imidazole). Then, a total of 12 fractions were collected. All the fractions were loaded onto a SDS PAGE gel to detect which fraction contains the eluted protein.

The fractions that contained the protein were collected and subjected to dialysis with a pore cut of MWCO 6 - 8,000 Da against PBS at 4°C (2 changes, 4 hours). The samples were pooled and stored at -70°C. The samples were ultracentrifuged at 100,000 ⨉ g for 1 hour (4°C) to discard possible aggregates. The soluble supernatant was aliquoted and stored at -70°C.

2.2.20.2. Immunisation of laying hens for antibody production

Laying hens have been used for the immunisation and preparation of antibodies. The extraction of IgY from the egg yolk can be performed by polyethylene glycol (PEG) precipitation (Polson et al., 1985; Polson et al., 1980).

The purified (His)10-tagged ARM56 was used to immunise laying hens. Immunisation of laying hens was performed in accordance to §10a of the German Animal Protection Law and registered with the Amt für Gesundheitlichen Verbraucherschutz, Behörde für Umwelt und Gesundheit, Freie und Hansestadt Hamburg. Therefore, 500 µg of recombinant protein were mixed 1:1 with the TiterMax^® Gold Adjuvant. Ten days after immunisation, the IgY was isolated to test the primary titer. Then, the hens were boostered and after another 10 days, the IgY was isolated to test the antibody titer.

2.2.20.3. Isolation of IgY

The egg yolk was separated from the egg white and it was washed with distilled water. The membrane of the egg yolk was removed and the egg yolk was transferred to a 50 mL reaction tube. Then 1 volume of potassium-phosphate-buffer (10 mM K-phosphate pH 7.2, 100 mM NaCl) was added to the egg yolk and it was incubated in a roller shaker to mix, followed by the addition of 1 volume of 7% PEG 6000 in potassium-phosphate-buffer and mixed. The samples were centrifuged at 16,000 ⨉ g for 10 min (4°C) and the supernatant was filtered through a gauze swab and paper filter. Next 10% PEG 6000 was added to the supernatant and left to dissolve, followed by centrifugation at 16,000 ⨉ g for 10 minutes (4°C). The supernatant was discarded and the pellet was solved in 10 mL potassium-phosphate-buffer and left to dissolve in a roller shaker for 30 minutes. Then 1 volume of 24% PEG 6000 in potassium-phosphate-buffer was added and mixed, followed by centrifugation at 16,000 ⨉ g for 10 minutes (4°C). The supernatant was discarded and 5 mL of potassium-phosphate-buffer was added to the pellet and left to dissolve in a roller shaker. The sample was centrifuged at 16,000 ⨉ g for 60 minutes (4°C) and the supernatant was transferred to a new reaction tube with 0.02% sodium azide.

2.2.20.4. Non-denaturing PAGE

In a native gel electrophoresis, protein mobility depends on the charge-to-mass ratio, physical shape and size of the protein. Non-denaturing protein extraction, non-denaturing gradient gel electrophoresis and Western blot were performed for isolation of oligomers and monomers. Cells in logarithmic growth were counted with a CASY^® counter and an equal of 2 ⨉ 10⁷ L. donovani promastigotes were sedimented (1250 ⨉ g, 8 min, 4°C), washed twice with 5 mL cold PBS and resuspended in 40 µL extraction buffer (15%

glycerol, 0.5 mM 1,10-phenanthroline, 10 mM Tris-HCl pH 8.0, 70 mM KCl). Samples were subjected to three cycles of freezing (liquid nitrogen) and thawing (block heater at 37°C) for three minutes each. Then a small amount (<10) of 2 mm ceramic beads (Ribolyser beads, Hybaid) were added to the tubes to break the DNA and it was shaken vigorously for 20 seconds. Cell lysates were subjected to centrifugation (16,000 ⨉ g, 10 min, 4°C) and

the supernatant, which contained the soluble protein fraction, was mixed (5:1, v/v) with loading buffer (50% glycerol, 0.1% bromophenol blue). The samples were electrophoretically separated on a 4 - 18% polyacrylamide (2.5 - 6% glycerol) gradient gel in 0.5 ⨉ Tris-borate-EDTA buffer (24 h, 20 V/cm, 4°C). Then, the gel was equilibrated at 60°C in transfer buffer (48 mM Tris, 39 mM glycine, 0.5% SDS, 20% methanol, 10 mM DTT) for 30 min, followed by Western transfer and immunological detection.

2.2.20.5. SDS-PAGE

SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) allows the separation of proteins according to their electrophoretic mobility. SDS is an anionic surfactant that denatures and disassociates proteins by binding to the polypeptide chain which gives an approximate distribution of charge per mass. PAGE is a technique that separates macromolecules according to their electrophoretic mobility. Proteins can be separated according to their length, conformation and charge. Using SDS-PAGE, proteins migrate through the gel matrix to the anode due to the negative charge given by the SDS molecules associated to the amino acids. Electrophoresis allows the determination of an approximate size of proteins when comparing their migration with defined molecular mass standards.

The lysates were loaded and separated on a discontinuous SDS-PAGE. Discontinuous gels were performed first by preparing the 10% resolving gel in 375 mM Tris-HCl (pH 8.8), 10% acrylamide/bis-acrylamide, 0.1% SDS, 0.1% APS and 0.1% TEMED. The gel solution was loaded between two glass plates of the electrophoresis chamber, followed by the addition of isopropanol. When the gel matrix was polymerised the isopropanol was discarded. Next, the 5% stacking gel that contained 125 mM Tris-HCl (pH 6.8), 5%

acrylamide/bis-acrylamide, 0.1% SDS, 0.1% APS and 0.1% TEMED was loaded followed by the comb which provides the wells. After polymerisation, the comb was removed and the chamber was filled with running buffer (25 mM Tris, 250 mM glycine, 0.1% SDS). The gel was running for 15 V cm^-1 for 1-2 hours. Two reference standards were used to identify the protein migration, an unstained protein marker in case of staining with Coomassie Blue and a prestained in case of being followed by Western transfer.

2.2.20.6. Coomassie Brilliant Blue staining

The Coomassie Brilliant Blue R-250 dye allows the examination of protein bands from gels after electrophoresis. The dye has a detection sensitivity of 0.5 µg cm² of protein present in a gel matrix. Protein gels were stained over night in staining solution (1 g L^-1 Coomassie Brilliant Blue R-250, 40% ethanol; 10% acetic acid) at room temperature. To detect the protein bands, gels were destained in destaining solution (40% ethanol; 10% acetic acid) for 2 hours at room temperature. When the protein bands were visible, the gel was washed twice with ddH2O and it was scanned to digitalise the image. The gel was dried between two layers of cellophane for conservation.

2.2.20.7. Semi-dry Western blot (Immunoblot)

Western blot is an analytical technique that allows the detection of specific proteins that are first separated in a gel matrix followed by transfer to a membrane, normally nitrocellulose or polyvinylidene difluoride (PVDF). The transfer of proteins was done using a PVDF membrane (Fuorotrans, 0.2 µm) equal to the size of the blot. The membrane was activated with methanol for 10 seconds and washed three times with ddH2O. The membrane was incubated shortly in blot-transfer-buffer with two pieces of 3MM Whatman paper. For the blotting, the Whatman paper was placed between the gel or membrane and the electrodes of the blotting chamber. The blotting conditions were determined by the size of the gel/membrane and the thickness of the gel. The proteins were transferred from the matrix gel to the membrane at 1 mA/cm² for 60 min with a maximum voltage of 30 V.

After Western transfer, the membranes were incubated overnight at 4°C or for 1 hour at room temperature with blocking solution (5% milk powder and 0.1% Tween 20 in Tris-buffered saline). Afterwards, the incubation with the first antibody (in blocking solution) was performed for 1 hour at room temperature. Then, the membrane was washed three times for 5 minutes with washing solution (0.1% Tween 20 in Tris-buffered saline), followed by the incubation with the second antibody-conjugated with AP (alkaline phosphatase) (in blocking solution) for 1 hour at room temperature. Then, the membrane was washed twice for 5 min with washing solution and the last incubation was done with the developing buffer. Then the blot was developed using the colorimetric detection of alkaline phosphatase activity. The substrates for the AP reaction were BCIP (5-bromo-4-chloro-3-indolyl-phosphate) and NBT (nitro blue tetrazolium). The reaction was done at room temperature until protein bands appeared, then the blot was washed twice with water and left to dry.

2.2.20.8. Trypsin digest

The trypsin digest was done in presence or absence of Triton X-100. Intact vesicles containing proteins are protected against the action of the trypsin. However, in presence of Triton X-100 the membranes are permeabilised and the trypsin can digest proteins.

Trypsin (EC 3.4.21.4) is a serine protease that digests proteins into peptides. It hydrolyses the peptide bonds at the carbonyl group of the amino acids lysine or arginine.

The verification of intact exosomes was performed in a modification of a published protocol (Bifeld et al., 2015; Silverman et al., 2010a). After ultra centrifugation, the intact exosomes were resuspended in a final volume of 75 µL 20 mM Tris-HCl (pH 7.4) and kept at 4°C overnight. The sample was splitted in three reaction tubes containing: i) 25 µL 20 mM Tris-HCl (pH 7.4); ii) 25 µL 20 mM Tris-Tris-HCl (pH 7.4) and 40 µg mL^-1 trypsin or iii) 25 µL in Tris-HCl (pH 7.4) with 0.1% Triton X-100 and 40 µg mL^-1 trypsin. The samples were then incubated at 37°C in a water bath for 2 hours. Trypsin was inactivated by adding PMSF at a final concentration of 22 mM. The sample was mixed vigorously for 10 seconds and proteins were precipitated by adding 4 vol of cold acetone for 2 hours at -20°C. The samples were centrifuged at 16.000 ⨉ g for 10 min at 4°C. The supernatant was discarded and the samples were air-dried. The samples were then subjected to SDS-PAGE and

Western transfer. The membranes were probed with the specific antibodies anti-ARM58 (1:200), anti-ARM56 (1:400), anti-HSP90 (1:500), and anti-HSL-U1 (1:1000). It was followed by incubation with the second antibodies anti-IgY-biotin (1:2000) and anti-IgG-biotin (1:2000). The last step included the incubation of the membranes with streptavidin-AP-conjugate (1:5000) to perform the colorimetric detection.